Nitrogen assimilation is of fundamental importance to the growth of plants. Of all the mineral nutrients required by plants, nitrogen is required in the greatest abundance. The main forms of nitrogen taken up by plants in the field are nitrate and ammonia, the principle components of nitrogenous fertilizers. Plants take up either nitrate or ammonium ions from the soil, depending on availability. Nitrate will be more abundant in well-oxygenated, non-acidic soils, whilst ammonium will predominate in acidic or waterlogged soils. Experiments on growth parameters of tobacco (Stöhr, 1999) clearly demonstrated that relative growth rate, chlorophyll content, leaf area and root area increased dramatically in response to increasing nitrate supply.
Roots take up nitrate and ammonia by the action of specific transporters, (Rothstein et al., 1998). In plants there are distinct transport systems that have different affinities for nitrate. The nitrate is then either reduced in the roots by the cytoplasmic enzyme nitrate reductase (NR) and enters the nitrogen assimilatory pathway, or it is transported to the shoots in the xylem. Nitrate is transported from the epidermal and cortical cells of the roots and into the vascular system to be transported to the shoots (Crawford, 1995). It enters the leaf via the apoplast and is transported across the plasma membrane into the mesophyll cells. Here it is either stored in vacuoles or reduced in the cytoplasm and enters the primary nitrogen assimilation pathway. When nitrate is present in excess it is stored in the vacuole. This serves both as an osmoticum and as a source of mineral N to be used when nitrate uptake is minimal (Crawford and Glass, 1998). The nitrate present in the cytoplasm is the starting point of primary nitrogen assimilation.
Nitrate is reduced in the cytosol by the cytoplasmic enzyme nitrate reductase (NR) to nitrite, which itself is rapidly reduced to ammonium by nitrite reductase (NiR) in the chloroplasts of leaves or in the plastids of non-photosynthetic organs (Crawford, 1995, Crete et al., 1997, Tobin and Bowsher, 2005). In the chloroplast the ammonium then enters the glutamine synthetase/glutamate synthase cycle (GS/GOGAT) where it is incorporated into the amino acid pool.
NR is considered to be the rate-limiting factor for growth and nitrate assimilation (Solomonson & Barber, 1990, Tischner, 2000) and is the first committed step of nitrogen assimilation. It catalyses the 2 electron reduction of nitrate to nitrite using NAD(P)H as an electron donor (Wray and Kinghorn, 1989).
There are 3 forms of NR; free NR (active), phosphorylated NR (active pNR), and pNR:14-3-3 (inactive). The ratio of the 3 forms is variable depending on external conditions (Kaiser et al., 2002). This complex regulation of NR controls the reduction of nitrate so that deleterious amounts of nitrite do not accumulate in the cell (Lillo et al., 2003). Lea et al., (2006) demonstrated that it is the post-translational regulation of NR in tobacco plants which has the greatest effect on NR activity and associated metabolite levels. Introducing NR using the constitutive promoter CaMV (35S) and thus deregulating NR at the transcriptional level had little effect on metabolite levels as the post-translational regulatory mechanism was still active. Loss of the post-translational control however, results in chlorosis of young tobacco leaves (Lillo et al., 2003). In tobacco, site-directed mutagenesis of Ser521 to aspartic acid prevents post-translational phosphorylation of NR (Kaiser et al., 2002, Lillo et al., 2003, Lea et al., 2006). When this post-translational control had been destroyed, constitutive activation of NR resulted in nitrite accumulation and chlorotic leaves.
A second function of NR is the reduction of nitrite to nitric and nitrous oxides. Nitric oxide (NO) is known to play an important signalling role in plant defence, growth and development (Wendehenne et al., 2004). NO production only uses 1% of the NR capacity and is dependant on nitrite concentration (Kaiser et al., 2001, Rockel et al., 2002). NO production from nitrite by purified maize NR was competitively inhibited by nitrate (50 μM) and the rate of NO production increased when nitrite levels accumulated as a result of higher nitrate reduction in relation to nitrite reduction (Rockel et al., 2002). In transgenic tobacco with a severe reduction in nitrite reductase (NiR) activity, a corresponding increase in NO emission was reported. This was also accompanied by an increased synthesis of 14-3-3 proteins involved in the regulation of nitrate reduction (Morot-Gaudry-Talarmain et al., 2002), and is likely to be related to attempts at controlling the potentially toxic accumulation of nitrite in the cell.
Nitrite reductase (NiR) is the second enzyme in the nitrate assimilation pathway and involves the transfer of 6 electrons from reduced ferredoxin to nitrite to form ammonium (Wray and Kinghorn, 1989). NiR from green leaves has a molecular mass of 63 kDa and is a monomer (Crété et al., 1997). NiR is found mainly in the chloroplasts of leaves from C3 plants, and in chloroplasts of mesophyll cells of C4 plants, as well as in the plastids of non-green tissues (Tobin and Bowsher, 2005).
The enzyme has been shown to be a metalloprotein (Swarmy et al., 2005) and contains the prosthetic sirohaem group, to which nitrite binds and a 4Fe/4S centre which is likely to be the initial electron acceptor.
NiR consists of 3 domains, folded compactly around the cofactors, sirohaem and a 4Fe/4S cluster. NiR forms a complex with its electron donor ferredoxin and substrate nitrite, the 4Fe/4S cluster receives the electrons from ferredoxin and transfers them to the sirohaem, which in turn transfers them to the substrate nitrite that remains bound until complete reduction to ammonia (Swamy et al., 2005).
NiR is encoded in the nucleus by the NiR gene (Dorbe et al., 1998), therefore the protein must be transported from the cytoplasm to the chloroplasts. The spinach NiR precursor protein is 32 amino acids longer than the mature protein. These additional amino acids are probably the transit peptide sequence directing the NiR to the chloroplast (Wray and Kinghorn, 1989) where this peptide must be cleaved to form the active protein.
Isoforms of NiR have been identified in a number of plants. In tobacco there are four NiR genes: NiR1 and NiR3 encode predominantly leaf specific NiRs and NiR2 and NiR4 encode mainly for root NiRs (Kronenberger et al., 1993, Stohr and Mack, 2001). Homologues of these genes have been found in two ancestral species of tobacco, NiR1 and NiR2 in Nicotiana tomentosiformis and NiR3 and NiR4 in Nicotiana sylvestris (Kronenberger et al., 1993).
There is only one NiR gene in A. thaliana, spinach and soybean, two in maize and hot pepper and three in wild oat (Wray and Kinghorn, 1989). The ratio of leaf to root mRNA of tobacco NiRs was found to be 3:1 (Kato et al., 2004), indicating that the leaf NiR plays a more significant role in nitrate assimilation. Kato et al. (2004) also demonstrated using quantitative RT-PCR that mRNAs for each of the 4 NiR genes were present in both leaves and roots but NiR2 and 4 only accounted for 10% of the total NiR mRNA in leaves. All four were induced after nitrate treatment.
Morot-Gaudry-Talarmain et al. (2002) produced antisense NiR tobacco plants with severe suppression of NiR activity. These plants showed drastically reduced growth and suggest that nitrite cytotoxicity in plants may be ascribed to the production of active nitrogen species such as NO (nitric oxide), N2O (nitrous oxide) and peroxynitrite, which in turn induces the nitration of tyrosine residues in proteins and phenolic ring structures. NO emission increased the synthesis of proteins such as 14-3-3's and cyclophilins.
NiR activity requires reduced ferredoxin as the electron donor, which is a product of photosynthesis (Tobin and Bowsher, 2005) and takes place in the chloroplast matrix. By isolating intact spinach chloroplasts, it was demonstrated that nitrite reduction can be triggered by illumination at a rate similar to that measured in the intact leaves. DCMU (3(3,4-dichlorophenyl)-1,1-dimethylurea) which interrupts the electron transport chain after PSII, and thus stops the availability of reduced ferredoxin, inhibited this reaction and showed that nitrite reduction is energetically directly linked/coupled to non-cyclic electron transport (Mohr and Schopfer, 1994).
In roots, nitrate assimilation takes place in leucoplasts. The reaction is similar to that which takes place in the chloroplast but is supplied by reduction equivalents (NADPH) via a ferredoxin-NADPH oxidoreductase enzyme from the oxidative pentose phosphate pathway (Tobin and Bowsher 2005).
The incorporation of ammonium into organic compounds is performed through the cyclic action of the enzymes glutamine synthetase (GS) and glutamine-2-oxoglutarate-aminotransferase (GOGAT) (Lea and Miflin, 1974). GS incorporates ammonium into glutamine (Gln) and glutamate (Glu) is derived from Gln by the action of GOGAT (Lea and Miflin, 2003; Glevarec et al., 2004). The process runs as a cycle, one of the glutamate molecules produced being used as a substrate by GS while the other is used for the synthesis of other amino acids. This pathway is of major importance as the Glu and Gln produced are donors for the biosynthesis of major N-containing compounds (Hodges, 2002).
Inputs to the cycle are ammonium, which can originate from several different sources, such as primary nitrate assimilation, photorespiration and nitrogen remobilisation (deaminating activity of glutamate dehydrogenase) and the substrate 2-oxoglutarate (2-OG) which could orginate from the isocitrate-dehydrogenases or amino transferases, but the exact route of 2-OG for ammonium assimiliation is still unclear (Hodges, 2002).
The resulting molecules generated by nitrogen assimilation, glutamine (Gln) and glutamate (Glu), are the nitrogen (N) donors for the synthesis of all the other amino acids and N-containing compounds in the cell, including nucleic acids, cofactors and chlorophyll. Therefore Glu and Gln are referred to as the pivotal amino acids and nitrate reduction and the GS/GOGAT cycle sit at the interface of the nitrogen and carbon (C) metabolism. N and C metabolism must be tightly co-ordinated, as the assimilation of N requires a supply of C skeletons in the form of 2-OG, as well as considerable ATP and reductant necessary for the reduction of nitrate to ammonium, and the incorporation of ammonium into Glu and Gln. This tight co-ordination has been highlighted in several studies by the strong correlation of N assimilation activities and metabolites with those of the photosynthetic carbon assimilation pathway (Martin et al., 2005). In tobacco, when plants were subjected to elevated CO2 levels, uptake of nitrate was enhanced by 7% (Kruse et al., 2002) and coincided with an increase in relative growth rates of 9%.
Nitrogen assimilation is also linked to sulphate assimilation. Sulphur interacts with nitrogen in such a way that a lack of one reduces the uptake and assimilation of the other (Hesse et al., 2004). Micro-array data from A. thaliana plants exposed to nitrate demonstrated expression of several sulphate transporters and assimilation genes in response to the nitrate treatment (Wang et al., 2003) in the same way that nitrate transporter and assimilation genes were upregulated. Indeed, both sulphite reductase (SiR) and NiR contain siroheme cofactors and iron-sulphur clusters which are essential for electron transfer. It is also known that SiR and NiR can reduce the substrate of the other enzyme but have much higher affinity for their own (Swamy et al., 2005). Therefore the activity of N assimilation, and particularly NiR, is dependant on the presence of sulphur (Swamy et al., 2005).
Nitrogen, mainly in the form of ammonium and amino acids, is also available to the plant via the pathways which recycle nitrogen, such as those achieved in photorespiration, senescence and amino acid catabolism. Photorespiration occurs when ammonium is released from glycine in the leaves of C3 plants during the conversion of glycine to serine by the mitochondrial enzyme glycine decarboxylase (GDC). The photorespiration pathway can lead to rates of ammonium assimilation of 10 times more than that of nitrate reduction especially when environmental conditions, such as drought, lead to stomatal closing and low carbon dioxide availability in the chloroplasts.
During senescence, the amino acids released following proteolysis are transaminated, so that the amino groups are transferred to Glu. An oxidative deamination reaction catalysed by glutamate dehydrogenase (GDH) is then able to liberate ammonium, 2-OG and reducing power (NADH). The ammonium can then act as a substrate for glutamine and asparagine synthesis and the 2-OG is metabolised in the Krebs cycle (Gleverac et al., 2004).
Theoretically GDH can also act in the aminating direction to synthesise glutamate from ammonium and 2-OG. The role of GDH in ammonium assimilation has been the subject of considerable controversy, which is still ongoing. However there is now considerable evidence that GDH functions predominantly in the deaminating direction in tissues with a low C/N ratio, that are converting amino acids into transport compounds, such as germinating seeds and senescing leaves (Miflin and Habash, 2002).
The regulation of NR and NiR activity is critical in controlling primary nitrogen assimilation throughout the plant and has a significant impact on the growth and development of the plant. However under certain conditions nitrate may accumulate, mainly in green photosynthetically active tissues, where it is stored in the vacuoles of the mesophyll cells. High levels of nitrate accumulation can occur during periods of low temperature and/or solar irradiation (for example, in greenhouse crops during the winter), when there is less photosynthetic capacity to assimilate the stored nitrate, or as a result of high nitrate levels in the soil. An increase in nitrate levels can have a number of deleterious consequences, not only in terms of plant growth but also in terms of human or animal health where the plant is consumed, as well as environmental consequences. Many of the adverse consequences of nitrate accumulation are mediated through the production of nitrite.
Nitrosamines form as part of a chemical reaction between a nitrosating agent and secondary or tertiary amine precursors. The source of nitrosating agents is nitrite which reacts with water to produce common nitrosating agents such as nitrous acid (HNO2), dinitrogen trioxide (N2O3) and peroxynitrite (ONOO−). This reaction is prevalent at elevated temperatures (such as cooking, smoking or drying processes) or in acidic conditions, such as in the stomach (Lee et al., 2006).
The formation of nitrosamines in the stomach is a result of endogenous nitrosation. Oral bacteria chemically reduce nitrate consumed in food and drink to nitrite, which can form nitrosating agents in the acidic environment of the stomach. These react with amines to produce nitrosamines and cause DNA strand breaks or cross linking of DNA.
Nitrosamines in tobacco are formed by microbial reduction of nitrate to nitrite which happens when the cells break down during senescence and curing, and the cell contents become available to micro-organisms which reside on the leaf Nitrosating agents derived from nitrite react with tobacco alkaloids to form tobacco-specific nitrosamines (TSNAs). Nitrite itself is formed during the leaf browning and stem drying phases. The amount of residual nitrate and nitrite in the leaf plays a major role in the reaction and the amount of TSNAs produced (Staff et al., 2005).
Nitrosamine compounds have been implicated in human cancers. This was first reported in 1956 by John Barnes and Peter Magee who demonstrated that dimethylnitrosamine (DMNA) induced liver tumours in rats. This led to the investigation of the carcinogenic properties of other nitrosamines with approximately 300 being tested and 90% found to be carcinogenic in a wide variety of experimental animals (Ellis et al., 1998). Human population studies have linked nitrosamines to cancers mainly of the oesophagus, oral cavity and pharynx (Isaacson, 2005).
Nitrate may be reduced to nitrite by micro-organisms arising from naturally occurring leaf flora, contamination (Isaacson, 2005) or from gut or oral bacteria (Ellis et al., 1998). As much as 25% of the nitrate ingested is taken up from the blood by the salivary glands to be excreted in the saliva. 20% of this is reduced to nitrite by the facultative anaerobes in the oral cavity which use nitrate as an alternative electron acceptor to oxygen in order to produce ATP. It is the nitrite which acts as the major nitrating agent, as nitrate itself will leave the body unmodified since nitrate cannot be metabolized by human enzymes.
Another problem associated with an excess of nitrate is the formation of methaemoglobin which gives rise to blue baby syndrome, where the oxygen carrying capacity of haemoglobin is blocked by nitrite, causing chemical asphyxiation in infants. Foetal haemoglobin, the predominant form in infants up to 3 months is oxidised more readily to methaemoglobin by nitrite than adult haemoglobin. Red blood cells contain methaemoglobin reductases that convert methaemoglobin back to haemoglobin, but the activity of this enzyme is half what it is in adults. Baby foods which contain vegetables, which are another source of increased nitrate content, are voluntarily measured to be less than 100 ppm and as spinach frequently exceeds this limit, products are often labelled not to be used in infants younger than 3 months (Greer et al., 2005).
As a consequence of these health concerns, a number of regulatory authorities have set limits on the amount of nitrate allowed in leafy green vegetables such as spinach and lettuce (e.g. European Commission Regulation 653/2003), depending on the time of harvest. These limits have resulted in any produce with a high nitrate content being unmarketable. Consequently there have been efforts to reduce nitrate content of plants by managing application of nitrogen-containing fertilisers or improved systems of crop husbandry (Isolovich et al., 2002). Some authorities have also set limits on the amounts of nitrate in drinking water.
An alternative method for modifying plant characteristics is through the use of genetic engineering techniques. The introduction and manipulation of specific coding sequences for targeted traits into plants in order to alter their physiology has proved successful for a number of crop species and model plants like tobacco, wheat, barley, A. thaliana and maize. The production of plants which contain herbicide resistant traits are commercially acceptable in some countries, particularly the USA, Spain and China. Crops containing traits which are of benefit to the consumer are also becoming available, such as golden rice (Syngenta) which has a phytoene synthase gene (from maize) and a carotene desaturnase gene (from Erwinia uredovara) inserted into its genome resulting in increased levels of vitamin A in the crop (Paine et al., 2005). Even so most genetic modification is used in the context of research as a tool to understand the function of specific genes within plants.
The soil bacterium Agrobacterium tumifaciens provides the tools for stable insertion of foreign genes into a plant and has been used in the transformation of many plant species, including tobacco, potato, tomato, A. thaliana, eucalyptus, etc. (Hoekema et al., 1983, Bendahmane et al., 2000). The A. tumifaciens naturally transfers its own plasmid DNA into plant genomes as a means of infecting the plant. A. tumifaciens contains a plasmid separate from the bacterial chromosome, known as the Ti plasmid. Within the Ti plasmid there is a region of DNA which can be transferred to the infected plant known as transfer-DNA (T-DNA). Also contained in the Ti plasmid are genes which facilitate the transfer of the T-DNA such as the vir region (a region which confers virulence for infection). A specific gene of interest (or genes) can be inserted into the transfer-DNA (T-DNA) of A. tumifaciens and this is then used to infect plants and generate transgenic populations.
As well as the gene of interest, a selectable marker gene is usually part of the T-DNA, such as neomycin phosphotranferase II (NPTII) which confers kanamycin antibiotic resistance to the plants expressing that gene, allowing a method of selection of transformed plants (Angenon et al., 1994). Agrobacterium-mediated transfer can result in more than one copy of the T-DNA being inserted into the plant genome. Multiple copies have been shown to lead to down-regulation of gene expression or gene silencing (Vaucheret et al., 1998, Han et al., 2004).
The regulation of NR protein in tobacco, potato and A. thaliana has been studied (for example Lea et al., 2006). NR sequences have been cloned and used in both over expression studies (Lea et al., 2004) and down-regulation studies (Lillo et al., 2004).
These studies have resulted in a further understanding of NR post-translational regulation which has been evolved by the plant to avoid the potential problems of nitrite accumulation. When NR was over-expressed or deregulated, nitrate levels were reduced throughout the day and night (Lea et al., 2006) and this caused a build-up of nitrite with ultimately damaging effects (Lillo et al., 2003, Lea et al., 2004). This is likely to be due to the fact that NiR is unable to reduce nitrite during the night in the leaf as the required reductant, reduced ferredoxin, is unavailable in the absence of photosynthesis.
An alternative approach was explored by Stitt et al., (1999), who used a mutant of tobacco with low NR activity, and observed an accumulation in nitrate content in the plant, which would also be undesirable. Contradictory to this, low NR activity in potato leaves resulted in the reduced nitrate levels in transgenic tubers (Djennane et al., 2002). In contrast, NiR has not been studied extensively. Takahashi et al. (2001) produced A. thaliana plants over-expressing spinach NiR and found that lines containing more than two copies of the transgene had low levels of mRNA. This phenomena of gene silencing can result from several different mechanisms employed by the plant, such as hyper-methylation of multiple copies which are integrated at one locus and co-suppression from RNAi mechanisms (Vaucheret et al., 1998, Han et al., 2004). Lines with one or two copies of the NiR gene showed significantly higher levels of 15N-labelled reduced nitrogen. This study focused on improving nitrate assimilation but did not investigate nitrate or nitrite levels in the transgenic plants.
Over-expression of the tobacco NiR genes in tobacco also resulted in a two-fold increase in NiR activity (Crété et al., 1997). However post-transcriptional regulation of tobacco leaf NiR expression was observed, since NiR activity and protein level were strongly reduced on ammonium-containing media despite constitutive expression of NiR mRNA. The effect of this on nitrate or nitrite levels was not reported.
Ozawa and Kawahigashi (2006) isolated a rice NiR gene and over-expressed it in a commercial rice variety (Koshihikari) for use as a selection system in the production of transformed rice. The introduction of NiR conferred good growth and regeneration ability of calli compared to the wild-type plants.
Accordingly, there is a need for a method for alleviating the adverse effects associated with nitrate and/or nitrite accumulation in plants. In particular, there is a need for a method for reducing nitrite content in plants, which may, for example, enhance nitrogen assimilation and/or reduce the toxicity of such plants to animals or humans.